VeriFiler Direct PCR Amplification Kit

Transcription

1 QUICK REFERENCE VeriFiler Direct PCR Amplification Kit Publication Number Rev. A Note: For safety and biohazard guidelines, refer to the Safety section in the VeriFiler Direct PCR Amplification Kit User Guide (Pub. no ). For every chemical, read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Product overview Prerequisites Treated paper substrates Swab substrates Human genomic DNA Perform PCR Perform electrophoresis Data analysis Limited Product Warranty Product overview The VeriFiler Direct Kit (Part no ) is a short tandem repeat (STR) multiplex PCR assay that amplifies 9 autosomal STR loci (D10S1248, D1S1656, D2S1338, D22S1045, D19S433, TH01, D2S441, D6S1043 and D12S391). It is optimized to allow direct amplification of the following types of single source samples: Blood samples on treated paper substrates without the need for sample purification. Buccal samples collected on treated paper substrates and treated with Prep n Go Buffer Buccal samples collected on swab substrates and treated with Prep n Go Buffer Genomic DNA Prerequisites Optimize the PCR cycle number for treated paper, untreated paper, and swab substrates (refer to the VeriFiler Direct PCR Amplification Kit User Guide). Treated paper substrates Sample prep guidelines Do not add water to the wells in the reaction plate before adding the punches. If your laboratory is experiencing static issues with the paper discs, you can: Blood samples: Prepare and dispense the 25 μl reaction mix into the wells of the reaction plate before adding the punches. Buccal samples: Add 2 μl of Prep n Go Buffer to the reaction plate prior to punching the sample. Make a 1.2 mm punch as close as possible to the center of the sample to ensure optimum peak intensity. Increasing the size of the punch may cause inhibition during PCR amplification. For manual punching: Place the tip of a 1.2 mm Harris Micro Punch on the card, hold the barrel of the Harris Micro Punch (do not touch the plunger), gently press and twist 1/4 turn, then eject the punch in to the appropriate well on the reaction plate. For automated punching: Refer to the User Guide of your automated or semi automated disc punch instrument for proper guidance. For Paternity Use Only. Not for use in Research or Clinical Diagnostic applications.

2 Prepare low-te buffer You can prepare the buffer as described below or order it from Teknova (Cat. no. T0223). To prepare low TE buffer: 1. Mix together: 10 ml of 1 M Tris HCl, ph ml of 0.5 M EDTA, ph ml glass distilled or deionized water Note: Adjust the volumes accordingly for specific needs. 2. Aliquot and autoclave the solutions. 3. Store at room temperature for appropriate period of time. Prepare the reactions 1. Add samples to the reaction plate: Well(s) Negative control Test samples Positive control IMPORTANT! Do not add a blank disc to the positive control well. Add the following to wells of a MicroAmp Optical 96-Well Reaction Plate mm blank disc 1.2 mm sample disc For 24 and 25 cycles 2 µl of DNA Control 007 For 26 and 27 cycles 1 µl of DNA Control 007 Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number. 2. Calculate the volume of each component needed to prepare the reactions using the table below. Reaction component Volume per reaction Master Mix 10.0 µl Primer Set 5.0 µl Low-TE Buffer 8.0 µl (Buccal samples on treated paper) or 10.0 µl (Blood samples on treated paper) Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! This kit has been optimized for a 25 μl PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles. 3. Prepare reagents. Thaw the Master Mix and the Primer Set, then vortex 3 seconds and centrifuge briefly before opening the tubes or bottles. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to8 C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 4. Pipet the required volumes of components into an appropriately sized polypropylene tube. 5. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 6. Dispense 25 μl for Blood samples and 23 μl for Buccal samples on treated paper substrates of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate. 2 VeriFiler Direct PCR Amplification Kit User Guide

3 7. Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film. IMPORTANT! If you are using the 9700 thermal cycler with a silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp compression pad (Cat. no ) on top of the plate to prevent evaporation during thermal cycling. The Veriti Thermal Cycler does not require a compression pad. 8. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 9. Amplify the samples in a Veriti 96 well Thermal Cycler or PCR System 9700 with the silver or gold plated silver 96 well block as described in Perform PCR on page 6. Swab substrates Sample prep guidelines For optimal performance, process swabs within 2 weeks of collection. Detach each buccal swab head from the swab shaft before lysis. Lysis is performed using Prep n Go Buffer (Cat. no for buccal swabs) and can be conducted in 1.5 ml tubes or in a 96 well deep well plate (Cat. no ) as described below. For optimum performance, lysis of a whole swab is recommended. To preserve the sample, evaluate lysis of a half swab. Prepare the sample lysate 1. Add 400 μl Prep n Go Buffer (Cat. no ) to 1.5 ml tubes or the appropriate wells of a 96 well deep well plate (Cat. no ). 2. Into each tube or well, put the entire head of each swab and let stand for 20 minutes at room temperature to lyse the sample. 3. After 20 minutes, transfer the sample lysate out of the sample plate into tubes or plates for storage, then discard the deep well plate containing the swab heads. Note: To minimize the risk of contamination, do not remove the swab heads from the sample lysate plate before transferring the lysate. 4. Proceed to the next section to prepare the reactions or see Store the sample lysate on page 4. Prepare the reactions 1. Add Prep n Go Buffer (Cat. no ) to the control wells in the reaction plate: Well(s) Negative control Positive control Add the following to wells of a MicroAmp Optical 96-Well Reaction Plate... 2 µl of Low-TE Buffer N/A 2. Calculate the volume of each component needed to prepare the reactions using the table below. Reaction component Volume per reaction Master Mix 10.0 µl Primer Set 5.0 µl Low-TE Buffer 8.0 µl Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! This kit has been optimized for a 25 μl PCR reaction volume to overcome the PCR inhibition expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the ability of the kit chemistry to generate full STR profiles. VeriFiler Direct PCR Amplification Kit User Guide 3

4 3. Prepare reagents. Thaw the Master Mix and the Primer Set, then vortex for 3seconds and centrifuge briefly before opening the tubes or bottles. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to8 C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 4. Pipet the required volumes of components into an appropriately sized polypropylene tube. 5. Vortex the reaction mix for 3 seconds, then centrifuge briefly. 6. Dispense 23 μl of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate. 7. Add samples to the reaction plate: Well(s) Add the following to wells of a MicroAmp Optical 96-Well Reaction Plate... Test samples 2 µl of sample lysate Positive control For 24 and 25 cycles 2 µl of DNA Control 007 For 26 and 27 cycles 1 µl of DNA Control 007 Note: The volumes of positive control are suggested amounts and may be adjusted if peak heights are too high or too low for your optimized cycle number. The final volume in each well is 25 μl (reaction mix and Prep n Go Buffer plus sample lysate or positive control). 8. Seal the plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film. IMPORTANT! If using the 9700 thermal cycler with silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp compression pad (Cat. no ) on top of the plate to prevent evaporation during thermal cycling. The Veriti Thermal Cycler does not require a compression pad. 9. Vortex the reaction mix at medium speed for 3 seconds. 10. Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders. 11. Amplify the samples in a Veriti 96 well Thermal Cycler or PCR System 9700 with the silver or gold plated silver 96 well block as described in Perform PCR on page 6. Store the sample lysate Cap the sample lysate storage tubes or seal the sample lysate storage plate with MicroAmp Clear Adhesive Film. Store the sample lysate as needed: If you are storing the sample lysate... Then place at... <2 weeks 2 to 8 C >2 weeks 15 to 25 C These storage recommendations are preliminary pending the results of ongoing stability studies. The effects of multiple freeze thaw cycles on the lysate have not been fully evaluated. Therefore, multiple freeze thaw cycles are not recommended. 4 VeriFiler Direct PCR Amplification Kit User Guide

5 Human genomic DNA Sample prep guidelines Extract genomic DNA from samples using an extraction method validated in your lab. Quantify genomic DNA from samples using a human DNA quantification method validated in your lab. Prepare low-te buffer In addition to the VeriFiler Direct Kit reagents, the use of low TE buffer (10 mm Tris, 0.1 mm EDTA, ph 8.0) is recommended. You can prepare the buffer as described in the procedure below or order it from Teknova (Cat. no. T0223). To prepare low TE buffer: 1. Mix together: 10 ml of 1 M Tris HCl, ph ml of 0.5 M EDTA, ph ml glass distilled or deionized water Note: Adjust the volumes based on your specific needs. 2. Aliquot and autoclave the solutions. 3. Store at room temperature for appropriate period of time. Prepare the reactions 1. Calculate the volume of each component needed to prepare the reactions, using the table below. DNA sample Volume per reaction VeriFiler Direct Master Mix 10.0 µl VeriFiler Direct Primer Set 5.0 µl Note: Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers. 2. Prepare reagents. Thaw the VeriFiler Direct Master Mix and the VeriFiler Direct Primer Set, then vortex the tubes for 3seconds and centrifuge them briefly before opening. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2 to8 C and, therefore, do not require subsequent thawing. Do not refreeze the reagents. 3. Pipet the required volumes of components into an appropriately sized polypropylene tube. 4. Vortex the reaction mix for 3seconds, then centrifuge briefly. 5. Dispense 15 μl of reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp tube. 6. Prepare the DNA samples: DNA sample To prepare... Negative control Add 10 µl of low-te buffer (10mM Tris, 0.1mM EDTA, ph 8.0). Test sample Positive control Dilute a portion of the test DNA sample with low-te buffer so that 2.0 ng of total DNA is in a final volume of 10 µl. Add 10 µl of the diluted sample to the reaction mix. Add 1 µl of DNA Control 007 (2.0 ng/µl) to provide 2.0 ng of total DNA in the positive control reaction. Add 9 µl of low-te buffer (10 mm Tris, 0.1 mm EDTA, ph 8.0) to get a final volume of 10 µl. The final reaction volume (sample or control plus reaction mix) should be 25 μl. 7. Seal the MicroAmp Optical 96 Well Reaction Plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film, or cap the tubes. 8. Centrifuge the tubes or plate at 3000 rpm for about 20 seconds in a tabletop centrifuge (with plate holders if using 96 well plates) to remove bubbles. VeriFiler Direct PCR Amplification Kit User Guide 5

6 9. Amplify the samples in a GeneAmp PCR System 9700 with the Silver 96 well block, or a GeneAmp PCR System 9700 with the Gold plated Silver 96 well block, or a Veriti 96 well Thermal Cycler. IMPORTANT! The VeriFiler Direct Kit is not validated for use with the GeneAmp PCR System 9700 with the Aluminium 96 well block. Use of this thermal cycling platform may adversely affect the performance of the VeriFiler Direct Kit. Perform PCR IMPORTANT! The VeriFiler Direct Kit is validated for use with the Veriti 96 well Thermal Cycler (Cat. no ) and the GeneAmp PCR System 9700 with the silver block (Cat. no. N ) or the gold plated silver block (Cat. no ). It is not verified for use with the Veriti 96 Well Fast Thermal Cycler (Cat. no ) or the GeneAmp PCR System 9700 with the aluminium block (Cat. no ). 1. Program the thermal cycling conditions. When using the GeneAmp PCR System 9700 with either 96 well silver or gold plated silver block, select the 9600 Emulation Mode. When using the Veriti 96 Well Thermal Cycler, refer to the following document for instructions on how to configure the Veriti instrument to run in the 9600 Emulation Mode: User Bulletin: Veriti 96 Well Thermal Cycler AmpFlSTR Kit Validation (Pub. no ). Initial incubation step Denature Optimum cycle number Anneal Final extension Final hold HOLD CYCLE HOLD HOLD 95 C 11 min 94 C 20 sec 59 C 3 min 60 C 15 min 4 C Determine the optimum cycle number for your laboratory according to the instructions in the user guide. For genomic DNA samples, set to 27 cycles and final extension conditions of 10 minutes at 60 C. IMPORTANT! The optimum conditions for the amplification of genomic DNA with the VeriFiler Direct Kit are 27 cycles of amplification with a 2 ng input DNA concentration. Perform internal validation studies to evaluate kit performance at each cycle number intended for operational use. 2. Load the plate into the thermal cycler and close the heated cover. 3. Start the run. 4. On completion of the run, store the amplified DNA. If you are storing the amplified DNA... Then place at... <1 weeks 2 to 8 C >1 weeks 15 to 25 C IMPORTANT! Protect the amplified products from light. IMPORTANT! The signal strength of the VIC channel artifact increases with storage of the amplification plate at 4 C, most commonly when plates are left at 4 C for a few days. We recommend storing amplification products at 20 C. Note: We did not validate using blood and buccal samples on untreated paper sample types. However, the VeriFiler Direct Kit can be used to process these sample types. Users may follow the protocol described in the Identifiler Direct Kit User Guide for performing internal validation studies for these sample types. 6 VeriFiler Direct PCR Amplification Kit User Guide

7 Perform electrophoresis Allelic ladder requirements To accurately genotype samples, you must run an allelic ladder sample along with the unknown samples. Instrument Number of allelic ladders to run One injection equals Number of samples per allelic ladder(s) per 4 injections 4 samples 15 samples + 1 allelic ladder 3130xl 1 per injection 16 samples 15 samples + 1 allelic ladder per 3 injections 8 samples 23 samples + 1 allelic ladder 3500xL 1 per injection 24 samples 23 samples + 1 allelic ladder IMPORTANT! Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between both single and multiple capillary runs (with larger size variations seen between samples injected in multiple capillary runs). We recommend the above frequency of allelic ladder injections, which should account for normal variation in run speed. However, during internal validation studies, verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment. It is critical to genotype using an allelic ladder run under the same conditions as the samples, because size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions. Electrophoresis instrument requirements Genetic Analyzer 3130/ 3130xl Operating System Windows XP Data Collection Software xL 1.0 Windows XP or Windows Vista Run modules and conditions 3.0 HIDFragmentAnalysis36_POP4_1 Injection conditions: 3130 = 3 kv/5 sec 3130xl = 3 kv/10 sec Dye Set G5 HID36_POP4 Injection conditions: 1.2kV/15 sec Dye Set G5 HID36_POP4 Injection conditions: 1.2kV/24 sec Dye Set G5 References Applied Biosystems 3130/3130xl Genetic Analyzers Using Data Collection Software v3.0, Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products User Bulletin (Pub. no ) Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Pub. no ) 3500 and 3500xL Genetic Analyzers Quick Reference Card (Pub. no ) We conducted validation studies for the VeriFiler Direct Kit using the 3130xl configurations. We conducted validation studies for the VeriFiler Direct Kit using the 3130xl configuration or the 3500xL configuration. VeriFiler Direct PCR Amplification Kit User Guide 7

8 Perform electrophoresis Prepare the samples for electrophoresis immediately before loading. 1. Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples: Reagent Volume per reaction GeneScan 500 LIZ Size Standard (31xx only) 0.5 µl or GeneScan 600 LIZ Size Standard v2.0 Hi-Di Formamide 9.5 µl Note: Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results. 2. Pipet the required volumes of components into an appropriately sized polypropylene tube. 3. Vortex the tube, then centrifuge briefly. 4. Into each well of a MicroAmp Optical 96 Well Reaction Plate, add: 10 μl of the formamide:size standard mixture 1μL of PCR product or Allelic Ladder Note: For blank wells, add 11 μl of Hi Di Formamide. 5. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 6. Heat the reaction plate in a thermal cycler for 3 minutes at 95 C. 7. Immediately place the plate on ice for 3 minutes. 8. Prepare the plate assembly on the autosampler. 9. Start the electrophoresis run. Data analysis To set up the GeneMapper ID X Software for data analysis, see the VeriFiler Direct PCR Amplification Kit User Guide (Pub. no ), the GeneMapper ID X Software v1.4 New Features and Installation Procedures User Bulletin (Pub. no ), or the GeneMapper ID X Software Version 1.0 Getting Started Guide (Pub. no ). Limited Product Warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologiesʹ General Terms and Conditions of Sale found on Life Technologies website at If you have any questions, please contact Life Technologies at The information in this guide is subject to change without notice. DISCLAIMER: LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/ or its affiliate(s) or their respective owners. Windows and Windows Vista are registered trademarks of Microsoft Corporation. Headquarters 5791 Van Allen Way Carlsbad, CA USA Phone Toll Free in USA For support visit lifetechnologies.com/support lifetechnologies.com March 2013